This is a continuing study of the mechanisms of 3 pyridine nucleotide-disulfide oxidoreductases: lipoamide dehydrogenase, glutathione reductase and thioredoxin reductase. Each of these flavoproteins contains an oxidation-reduction active cystine residue. There is a base in the active center which is protonated upon 2-electron reduction forming an ion pair with thiolate. The general principal of activation of thiols by ion pair formation will be explored in other enzymes. Chemical modification of the base and other active center residues will be attempted. These studies will be extended to thioredoxin reductase. The steady state and rapid reaction kinetics of thioredoxin reductase will be reinvestigated in an effort to assess the relative importance of EH2 and EH4 in catalysis. The charge transfer absorption characteristic of 2-electron reduced lipoamide dehydrogenase and glutathione reductase if pH dependent, and at low pH the charge transfer complex is not the only species present. The absorbance and fluorescence of lipoamide dehydrogenase and glutathione reductase as a function of pH are being investigated. Certain features of the gross structure of lipoamide dehydrogenase will be compared with the emerging structure of human erythrocyte glutathione reductase (sequence and X-ray structure in progress elsewhere), e.g., is the active center disulfide in the same portion of the polypeptide chain? The spectral properties of human erythrocyte glutathione reductase will be examined in reductive titrations. A histidine residue and a tyrosine residue have been shown to be in the active site of D-amino acid oxidase; peptides containing these residues are being isolated.